Altered expression of proteoglycans in E1A-immortalized rat fetal intestinal epithelial cells in culture.

Normal and E1A-immortalized rat fetal intestinal epithelial SLC-11 cells were compared for the characteristics of the 35S-labeled proteoglycans isolated from their cell-associated and secreted fractions. In comparison with control cells in primary culture, immortalized SLC-11 cells: (a) secreted larger amounts of radiolabeled proteoglycans; (b) contained larger amounts of membrane-intercalated proteoglycans, analyzed by hydrophobic affinity chromatography on octyl-Sepharose; (c) produced cell-associated and secreted proteoglycans of smaller hydrodynamic size, assessed by measurement of Kav values; (d) contained a higher percentage of heparan sulfate in the cell-associated proteoglycans, determined by differential susceptibility of glycosaminoglycans to specific glycosaminoglycan lyases; (e) displayed heparan sulfate and chondroitin sulfate with a shorter chain length; and (f) synthesized glycosaminoglycans with a lower degree of sulfation, determined by ion-exchange chromatography. Taken together, these results demonstrate that in E1A-immortalized intestinal epithelial SLC-11 cells, the expression of proteoglycans alters considerably at an early stage of oncogenic transformation.

Increased protein kinase C alpha expression in human colonic Caco-2 cells after insertion of human Ha-ras or polyoma virus middle T oncogenes.

The proteins encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancers. To investigate the mechanism(s) whereby oncogenic p21ras and pp60c-src contribute to malignant transformation of intestine, human colonic Caco-2 cells transfected with an activated (Val 12) human Ha-ras gene (Caco-2-T cells) or Py-MT oncogene, a constitutive activator of pp60c-src tyrosine kinase activity (Caco-2-MT cells), were analyzed for tumorigenicity, protein kinase C (PKC) isoform expression, and PKC activity. As compared with control vector Caco-2-H cells, Caco-2-T and Caco-2-MT cells displayed: (a) an enhanced tumorigenicity in nude mice; (b) a 4-fold increase in the level of PKC-alpha mRNA which was not due to enhanced mRNA stability and was mediated through a PKC-independent pathway since it persisted after PKC depletion; (c) increased PKC-alpha immunoreactive protein content (3-fold), total PKC catalytic activity (3.5-fold), and total cell number of [3H]phorbol-12,13-dibutyrate binding sites (4-fold); and (d) a 1.7-fold higher membrane-bound/total PKC activity ratio together with 1.8- and 1.5-fold increases in [3H]arachidonate- and [3H]myristate-labeled diacylglycerol levels. In conclusion, the tumorigenic progression induced by oncogenic p21ras or the Py-MT/pp60c-src complex in Caco-2 cells is associated with increased PKC-alpha gene transcription and PKC-alpha expression as well as with constitutive PKC activation. These results provide the first evidence that the PKC-alpha gene is a target for the signaling pathways of oncogenically activated p21ras and pp60c-src in human colonic cells. They raise the possibility that PKC-alpha is an effector of these oncoproteins for activation of Caco-2 cell tumorigenic potential.

Down-regulation of NF-kappaB activity and NF-kappaB p65 subunit expression by ras and polyoma middle T oncogenes in human colonic Caco-2 cells.

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras or pp60c-src in human colonic cells is associated with alterations of the activity and expression of nuclear factor kappaB (NF-kappaB), a transcription factor suspected to participate in the development of cancer. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated Val-12 human Ha-ras gene or with the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines exhibited: (i) decreased constitutive NF-kappaB DNA-binding activity and NF-kappaB-mediated reporter gene expression, without alteration of their response to TNF-alpha for activation of these parameters; (ii) reduced NF-kappaB cytosolic stores along with a decreased p65 expression due, at least in part, to destabilization of p65 mRNA; (iii) a decrease in adhesion to extracellular matrix component-coated substrata which was partially corrected when stimulating NF-kappaB transcriptional activity with TNF-alpha. These results indicate that the tumorigenic progression induced by oncogenic p21ras or PyMT/pp60c-src in human colonic Caco-2 cells is associated with a down-regulation of p65 expression and NF-kappaB activity which could be responsible for the reduced adhesive properties of these cells after oncogene transfection.

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras.

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras Constitutive activation of the ras proto-oncogene is a frequent and early event in colon cancers, but the downstream nuclear targets are not fully understood. The Cdx-1 and Cdx-2 homeobox genes play crucial roles in intestinal cell proliferation and differentiation. In addition, Cdx-2 is a colonic tumour-suppressor gene, whereas Cdx-1 has oncogenic potential. Here, we show that constitutive activation of ras alters Cdx-1 and Cdx-2 expression in human colonic Caco-2 and HT-29 cells that harbour a normal ras proto-oncogene. Oncogenic ras downregulates Cdx-2 through activation of the PKC pathway and a decline in activity of the Cdx-2 promoter AP-1 site. This decline results from a PKC-dependent decrease in the relative expression of c-Jun, an activator of Cdx-2 transcription, compared to c-Fos, an inhibitor of Cdx-2. Unlike Cdx-2, Cdx-1 is upregulated by oncogenic ras and this effect is mediated by activation of the MEK1 pathway. These results indicate that oncogenic ras activation has opposite effects on Cdx-1 and Cdx-2 expression through distinct signalling pathways and they provide the first evidence for a functional link between ras activation and the downregulation of the Cdx-2 tumour-suppressor gene in colon cancer cells.

Oncogene-induced up-regulation of Caco-2 cell proliferation involves IGF-II gene activation through a protein kinase C-mediated pathway.

We previously reported that ras and polyoma middle T (PyMT), a constitutive activator of the src protooncogene product, up-regulated Caco-2 cell proliferation along with protein kinase C (PKC) alpha expression and PKC activity. We aimed to investigate whether oncogene-induced up-regulation of Caco-2 cell proliferation involved stimulation of the autocrine IGF-II/IGF-I receptor (IGFIR) loop described in these cells and if so, to analyse the role of overexpressed and activated PKC. Compared with control vector transfected Caco-2 cells, ras- and PyMT-transfected cells exhibited increased expression of the 6.0 and 4.8 kb IGF-II transcripts. This was due to increased activity of the P3 and P4 promoters of the IGF-II gene which correlated with increased expression and DNA-binding activity of Sp1, a transcription factor interacting with several specific sites in P3 and P4 promoters. Oncogene-transfected cells displayed enhanced autocrine IGF-II production, which was fully responsible for the oncogene-induced increase in their proliferation since this increase was blunted by anti-human IGF-II and IGF1R (alphaIR3) antibodies. PKC mediated oncogene activation of the IGF-II gene presumably through action on Sp1 since (i) PKC activation by phorbol 12-myristate 13-acetate increased Sp1 expression, P3 and P4 activity and IGF-II mRNA in control but not in oncogene-transfected cells; and (ii) PKC inhibition by the PKC inhibitor Gö6976 reduced Sp1, P3 and P4 activity and IGF-II mRNA in all three cell lines. This is the first evidence that ras- and PyMT/src oncogenes up-regulate Caco-2 cell proliferation through a PKC-mediated pathway which stimulates IGF-II gene transcription and thereby increases autocrine IGF-II production. The mechanisms underlying IGF-II gene activation by PKC most probably involve action on Sp1.

Insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades involving GSK-3beta inhibition and Ras activation.

We examined the interplay between the insulin/IGF-1- and beta-catenin-regulated pathways, both of which are suspected to play a role in hepatocarcinogenesis. Insulin and IGF-1 stimulated the transcription of a Lef/Tcf-dependent luciferase reporter gene by 3-4-fold in HepG2 cells. This stimulation was mediated through the activation of phosphatidylinositol 3-kinase (PI 3-K)/Akt and the inhibition of glycogen synthase kinase-3beta (GSK-3beta) since the effects of insulin and IGF-1 were inhibited by dominant-negative mutants of PI 3-K or Akt and an uninhibitable GSK-3beta. Together with inhibiting GSK-3beta, insulin and IGF-1 increased the cytoplasmic levels of beta-catenin. The PI 3-K/Akt/GSK-3beta pathway was not the sole to mediate insulin and IGF-1 stimulation of Lef/Tcf-dependent transcription. The Ras signalling pathway was also required as (i) the stimulatory effects of insulin and IGF-1 were inhibited by dominant-negative Ras or the MEK1 inhibitor PD98059 and (ii) activated Ha-Ras or constitutively active MEK1 synergized with catalytically inactive GSK-3beta to stimulate Lef/Tcf-dependent transcription. This study provides the first evidence that insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades bifurcating downstream of PI 3-K and involving GSK-3beta inhibition and Ras activation. These findings demonstrate for the first time the ability of insulin and IGF-1 to activate the beta-catenin pathway in hepatoma cells and thereby provide new insights into the role of these factors in hepatocarcinogenesis.

Smad7 inhibits the survival nuclear factor kappaB and potentiates apoptosis in epithelial cells.

In this study, we examined the effect of the stable expression of Smad7 in two different cell lines on apoptosis induced by various stimuli including TGF-beta, serum withdrawal, loss of cell adhesion (anoikis) and TNF-alpha. Smad7 increased TGF-beta-mediated apoptosis in Mv1Lu cells as well as anoikis and/or serum withdrawal-induced apoptosis in Mv1Lu and MDCK cells. Smad7 markedly decreased the activity of the survival NF-kappaB transcription factor in MDCK cells. Interestingly, the stable expression of oncogenic Ras in MDCK cells which suppressed Smad7 inhibition of NF-kappaB also suppressed Smad7 potentiation of serum withdrawal-induced apoptosis and anoikis. In addition, Smad7 inhibited TNF-alpha stimulation of NF-kappaB and increased TNF-alpha-mediated apoptosis in MDCK cells. Our results provide the first evidence that Smad7 induces sensitization of cells to different forms of cell death. They moreover demonstrate that Smad7 inhibits the survival NF-kappaB factor, providing a potential mechanism whereby Smad7 potentiates cell death.

The lipid composition of plasma membrane and mitochondrial fractions from epididymal adipocytes of cold-acclimated rats.

1. The effects of cold acclimation (5 degrees C) on the lipid composition of plasma membrane and mitochondrial fractions from epididymal adipocytes of rats were studied. 2. The adipocyte plasma membrane fraction of the cold-acclimated rats had lower lipid, phospholipid and cholesterol to protein weight ratios, a lower cholesterol to sphingomyelin molar ratio, and a higher linoleic acid content in the phospholipids than controls. 3. The mitochondrial fraction of the cold-acclimated rat adipocyte had lower ratios of cholesterol to protein (weight), to phospholipid and to cardiolipin (molar), and less sphingomyelin content than did controls. 4. These data, discussed in terms of alterations in physical and biochemical properties, indicate cold-induced changes at the membrane level in rat epididymal adipocytes.

Insulin receptor autophosphorylation sites tyrosines 1162 and 1163 control both insulin-dependent and insulin-independent receptor internalization pathways.

We previously reported that Chinese hamster ovary (CHO) cell lines overexpressing mutated human insulin receptors (hIRs) in which the tyrosine residues 1162 and 1163 were replaced by phenylalanines (CHO-Y2) exhibited a marked defect in hormone-induced receptor internalization as compared to CHO transfectants overexpressing wild-type hIRs (CHO-R). These two cell lines are now used to compare the role of tyrosines 1162-1163 in basal and ligand-stimulated receptor internalization as well as in receptor turnover. We show here that (1) in CHO-Y2 cells, basal endocytosis, like insulin-induced internalization, was markedly altered despite normal receptor turnover and (2) in both CHO-R and CHO-Y2 cells, basal receptor endocytosis was altered by tunicamycin, an inhibitor of protein N-glycosylation, whereas insulin-induced internalization was not. These results support a role for tyrosines 1162-1163 of the IR beta-subunit major autophosphorylation domain in both basal and ligand-stimulated receptor endocytosis and provide evidence that the two processes follow distinct pathways.

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization.

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.

Insulin stimulation of glucose metabolism in rat adipocytes: possible implication of protein kinase C.

The hypothesis that insulin stimulation of glucose and lipid metabolism in rat adipocytes may involve the activation of protein kinase C was evaluated. 4 beta-Phorbol 12 beta-myristate, 13 alpha-acetate (PMA, 0.1-1000 ng/ml), a potent tumor promoter acting as a substitute for diacylglycerol to activate protein kinase C, stimulated 2-deoxyglucose transport in a time- and dose-dependent manner, without affecting passive glucose diffusion. PMA (0.1-1000 ng/ml) also elicited a dose-dependent activation of lipogenesis from [3-3H] glucose. Maximal PMA effects (100 ng/ml) on both processes were 60% of insulin maximal effects. In contrast, PMA (1-1000 ng/ml) failed to mimic the ability of insulin to stimulate lipogenesis from [3H]acetate. 4 beta-Phorbol 12,13 dibutyrate, mezerein, 1-oleyl-2-acetyl-glycerol, 1,2 diolein, known as protein kinase C activators, also markedly stimulated glucose metabolism whereas 4 alpha-phorbol 12,13 didecanoate and 4 beta-phorbol 13-monoacetate, shown not to activate protein kinase C, were ineffective. Mellitin, a cytotoxin-inhibiting protein kinase C, markedly decreased both PMA and insulin stimulation of glucose metabolism. PMA decreased insulin submaximal effects on 2-deoxyglucose transport without inhibiting insulin binding. Maximal PMA and insulin effects on 2-deoxyglucose transport and lipogenesis from [3-3H]glucose were not additive. Both PMA and insulin activated each metabolic process in a calcium-dependent manner. PMA, like insulin, no longer stimulated 2-deoxyglucose transport in fat cells treated with 2,4-dinitrophenol. These data show that PMA exhibited specific insulin-like properties on glucose metabolism in fat cells, without any effect on lipid synthesis from acetate. They indicate that PMA and insulin bioeffects may share a common step implicating a calcium- and energy-dependent process, distal to the initial insulin binding event. Our results suggest that protein kinase C may play a role in insulin regulation of glucose metabolism.

Decreased insulin responsiveness in fat cells rendered protein kinase C-deficient by a treatment with a phorbol ester.

Insulin stimulation of 2-deoxyglucose transport and lipogenesis from glucose was examined in fat cells in which protein kinase C had been down-modulated by a 3 h pretreatment with 5 X 10(-7) M 4 beta-phorbol 12 beta-myristate, 13 alpha-acetate (PMA). As compared to control fat cells, the down-modulated cells exhibited a 55-65% decrease in insulin responsiveness with no change in either the hormone sensitivity or the insulin receptor affinity. The present study shows that fat cells made protein kinase C-deficient by chronic treatment with PMA exhibit an insulin-resistant state, distal to the initial step of hormone binding.

Insulin induction of protein kinase C alpha expression is independent of insulin receptor Tyr1162/1163 residues and involves mitogen-activated protein kinase kinase 1 and sustained activation of nuclear p44MAPK.

We examined the effect of insulin on protein kinase C alpha (PKCalpha) expression and the implication of the mitogen-activated protein kinase kinase 1 mitogen-activated protein kinase (MAPK) pathway in this effect. PKCalpha expression was measured by quantitative RT-PCR and Western blotting using Chinese hamster ovary (CHO) cells overexpressing human insulin receptors of the wild type (CHO-R) or insulin receptors mutated at Tyr1162/1163 autophosphorylation sites (CHO-Y2). In CHO-R cells, insulin caused a time- and concentration-dependent increase in PKCalpha messenger RNA, with a maximum at 6 h and 10-(8)M insulin. This increase involved a transcriptional mechanism, as it was not due to stabilization of PKCalpha messenger RNA and was associated with a similar increase in the immunoreactive PKCalpha level. Insulin induction of PKCalpha expression involved the MEK1MAPK pathway, as it was 1) almost completely suppressed by the potent MEK1 inhibitor PD98059, 2) mimicked by the dominant-active MEK1 (S218D/S222D) mutant, and 3) associated with sustained MAPK activation. In CHO-Y2 cells in which the early phase of MAPK activation by insulin was lost and only the late and sustained phase of activation was observed, insulin signaling of PKCalpha expression was preserved and again involved the MEK1-MAPK pathway. Moreover, we show that in both CHO-R and CHO-Y2 cells, insulin stimulation of PKCalpha gene expression was associated with prolonged activation of nuclear p44MAPK. These results indicate that induction of PKCalpha gene expression by insulin is independent of Tyr1162/1163 autophosphorylation sites and correlates with sustained activation of p44MAPK at the nuclear level.

Increased insulin action in cultured hepatocytes from rats with diabetes induced by neonatal streptozotocin.

Previous studies have shown that Wistar rats injected at birth (n0) with STZ (n0-STZ) develop as adults a noninsulin-dependent diabetic state characterized by a lack of insulin response to glucose in vivo, a mild basal hyperglycemia, and an impaired glucose tolerance. Our former in vivo studies using the insulin-glucose clamp technique revealed an increased insulin action upon hepatic glucose production in these animals. We have now cultured hepatocytes from these mildly diabetic rats in parallel with hepatocytes from control rats, to examine more closely basal and insulin-regulated glucose production and glucose incorporation into glycogen. In addition, we extended our investigation to other hepatic functions such as lipid synthesis and amino acid transport, which could not be studied in vivo. Although glucose production from glycogenolysis or gluconeogenesis in absence or presence of glucagon was identical in the two cell populations, glucagon-stimulated glycogenolysis was more sensitive to insulin action in diabetic hepatocytes. Similarly, insulin action on glucose incorporation into glycogen, lipogenesis, and amino acid transport were enhanced in diabetic hepatocytes. The hormone effect was manifested by an increase in the sensitivity and/or in the responsiveness, reflecting the multiplicity of the pathways whereby the insulin signal is transduced through the insulin receptor to multiple postreceptor sites. To gain insight into the possible mechanism of these disturbances, we evaluated the initial insulin receptor interaction and the kinase activity of the receptor beta-subunit. In accordance with our previous study on intact livers, we found no alteration in either of these parameters in n0-STZ rat hepatocytes. Thus, the present study clearly demonstrates that these diabetic rats exhibit a postreceptor hyperresponsiveness to insulin at the cellular level. It strengthens the notion that a beta-cell deficiency with glucose intolerance does not necessarily lead to a hepatic insulin resistance.

Insulin-mediated cell proliferation and survival involve inhibition of c-Jun N-terminal kinases through a phosphatidylinositol 3-kinase- and mitogen-activated protein kinase phosphatase-1-dependent pathway.

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.

Antiinsulin receptor autoantibodies induce insulin receptors to constitutively associate with insulin receptor substrate-1 and -2 and cause severe cell resistance to both insulin and insulin-like growth factor I.

We report here that antiinsulin receptor (anti-IR) autoantibodies (AIRs) from a newly diagnosed patient with type B syndrome of insulin resistance induced cellular resistance not only to insulin but also to insulin-like growth factor I (IGF-I) for the stimulation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase activities and of glycogen and DNA syntheses. The molecular mechanisms of this dual resistance were investigated. Patient AIRs bound the IR at the insulin-binding site and caused insulin resistance at the IR level by inducing a 50% decrease in cell surface IRs and a severe defect in the tyrosine kinase activity of the residual IRs, manifested by a loss of insulin-stimulated IR autophosphorylation and IR substrate-1 (IRS-1)/IRS-2 phosphorylation. In contrast, cell resistance to IGF-I occurred at a step distal to IGF-I receptors (IGF-IRs), as AIRs altered neither IGF-I binding nor IGF-I-induced IGF-IR autophosphorylation, but inhibited the ability of IGF-IRs to mediate tyrosine phosphorylation of IRS-1 and IRS-2 in response to IGF-I. Coimmunoprecipitation assays showed that in AIR-treated cells, IRs, but not IGF-IRs, were constitutively associated with IRS-1 and IRS-2, strongly suggesting that AIR-desensitized IRs impeded IGF-I action by sequestering IRS-1 and IRS-2. Accordingly, AIRs had no effect on the stimulation of mitogen-activated protein kinase activity or DNA synthesis by vanadyl sulfate, FCS, epidermal growth factor, or platelet-derived growth factor, all of which activate signaling pathways independent of IRS-1/IRS-2. Thus, AIRs induced cell resistance to both insulin and IGF-I through a novel mechanism involving a constitutive and stable association of IRS-1 and IRS-2 with the IR.

Enterocytic differentiation of the human Caco-2 cell line is correlated with down-regulation of fibronectin and laminin.

Human intestinal Caco-2 cells were used to examine the expression of fibronectin (FN) and laminin (LN) during enterocytic differentiation. Combination of immunoprecipitation, Western and Northern blotting revealed that Caco-2 cells expressed a classical FN and a variant form of LN: besides B1 and B2 chains, LN contained a 350-kDa heavy chain instead of the 400-kDa A chain. Throughout Caco-2 cell differentiation, FN and LN synthesis decreased at both mRNA and protein levels. These data indicate that enterocytic differentiation involves both transcriptional and/or post-transcriptional down-regulation of FN and LN gene expression.

Phorbol ester-induced protein kinase C translocation and lysosomal enzyme release in normal and cystic fibrosis fibroblasts.

The ability of the tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to induce protein kinase C (PKC) translocation and lysosomal enzyme release was examined in skin fibroblasts from normal subjects and from patients with cystic fibrosis (CF). As compared to normal fibroblasts, those CF exhibited: (i) an increased sensitivity to the effect of PMA on the disappearance of PKC from cytosolic fractions as well as a greater and earlier recovery, in the membrane fraction, of the PKC activity lost in the cytosolic fraction; (ii) an earlier response to PMA for its effect on beta-N-acetylglucosaminidase release. In contrast, the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) proved ineffective in inducing PKC translocation and beta-N-acetylglucosaminidase release in both normal and CF fibroblasts. The data suggest a defect in the regulation of PKC activity in CF fibroblasts, which may lead to altered secretion.

Further characterization of the insulin receptor glycosidic moiety in rat adipocytes.

Simultaneous or sequential treatment of rat adipocytes with neuraminidase plus beta-galactosidase decreased insulin binding by 43%. No modification was observed with either enzyme individually. alpha-Mannosidase enhanced insulin binding (38%), whereas beta-N-acetylglucosaminidase and alpha-L-fucosidase were ineffective. Lectins that interact with galactose (Ricinus communis I, RCAI), mannose, Lens culinaris agglutinin (LCA), Concanavalin A (Con A) or N-acetylglucosamine (wheat-germ agglutinin, WGA) decreased insulin binding by 43, 57, 59 and 85% respectively. Lectin inhibition was dose-dependent, saturable and prevented by specific monosaccharides. RCAI, LCA, Con A and WGA decreased the insulin dissociation process by 45, 90, 78 and 84% respectively. Lectins specific for sialic acid, terminal galactose, N-acetylgalactosamine or fucose (Limulus polyphemus, peanut, soybean and Ulex I agglutinins) did not modify either insulin binding or dissociation. These results indicate involvement of penultimate D-galactose, internal N-acetyl-D-glucosamine and D-mannose residues in both processes. They suggest that, in rat adipocytes, a glycosidic moiety participates in the insulin-receptor interaction through N-linked oligosaccharides of the 'complex type'.

Carbohydrate determinants involved in both the binding and action of insulin in rat adipocytes.

The insulin receptor apparent affinity was markedly decreased in fat cells treated with lectins specific either for D-galactose (Ricinus communis agglutinin I, RCAI), D-mannose (concanavalin A, Con A, Lens culinaris agglutinin, LCA) or N-acetyl-D-glucosamine (wheat germ agglutinin, WGA), as indicated by a rightward shift of the binding competition curves and almost lineared Scatchard plots. Limulus polyphemus agglutinin (LPA), specific for sialic acid, was ineffective. All lectins enhanced 2-deoxy-D-glucose uptake with relative bioactivities (maximal lectin effect/maximal insulin effect) of 68-86%. Insulin and lectin stimulatory effects were antagonized by specific carbohydrates used as competitors and inhibited by cytochalasin B (70 microM). Maximal effects of insulin and lectins were not additive and were completely abolished in neuraminidase-treated fat cells. Lectins did not affect insulin degradation. These data show that sialylated glycosidic moieties containing D-galactose, D-mannose and N-acetyl-D-glucosamine units are involved in both processes of insulin 'high affinity' binding and activation of glucose transport but are not implicated in hormone degradation. They suggest that N-linked carbohydrate chains of the complex type may be essential for functional insulin receptor and post-receptor systems.